Methods for the identification of antibiotics that are not susceptible to antibiotic resistance in pseudomonas aeruginosa

ABSTRACT

The “instant evolution” system was initially developed in  E. coli , primarily because of the ease with which this organism can be genetically manipulated. Because many of the functionally important regions of rRNA are conserved among bacteria, drug leads developed against conserved targets in the  E. coli  system may produce broad-spectrum anti-infectives. In order to develop a system to produce narrow-spectrum anti-infectives, herein we disclose methods for identifying functional mutant  P. aeruginosa  ribosomes suitable as drug targets and for identifying drug candidates that do not bind to the human 16S rRNA.

GOVERNMENT SUPPORT

This invention was made with government support under NIH SBIR GrantNumber AI060275-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/654,911, filed Oct. 18, 2012, which is a divisional applicationof U.S. application Ser. No. 11/914,062, filed Jun. 30, 2008, which is aU.S. national stage application of PCT Patent Application No.PCT/US2006/018187, filed May 11, 2006, which claims the benefit of U.S.Application Ser. No. 60/680,134, filed May 11, 2005, the entire contentof each is hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which is identicalto the Sequence Listing submitted in the parent application Ser. No.11/914,062, filed on Jun. 30, 2008, via EFS-Web in ASCII format, and ishereby incorporated by reference in its entirety. The ASCII copy isentitled “WSS00401_SEQUENCE_LISTING.txt” and is 88012 bytes in size.

BACKGROUND OF THE INVENTION

Ribosomes are composed of one large and one small subunit containingthree or four RNA molecules and over fifty proteins. The part of theribosome that is directly involved in protein synthesis is the ribosomalRNA (rRNA). The ribosomal proteins are responsible for folding the rRNAsinto their correct three-dimensional structures. Ribosomes and theprotein synthesis process are very similar in all organisms. Onedifference between bacteria and other organisms, however, is the waythat ribosomes recognize mRNA molecules that are ready to be translated.In bacteria, this process involves a base-pairing interaction betweenseveral nucleotides near the beginning of the mRNA and an equal numberof nucleotides at the end of the ribosomal RNA molecule in the smallsubunit. The mRNA sequence is known as the Shine-Dalgarno (SD) sequenceand its counterpart on the rRNA is called the Anti-Shine-Dalgarno (ASD)sequence.

There is now extensive biochemical, genetic and phylogenetic evidenceindicating that rRNA is directly involved in virtually every aspect ofribosome function (Garrett, R. A., et al. (2000) The Ribosome:Structure, Function, Antibiotics, and Cellular Interactions. ASM Press,Washington, D.C.). Genetic and functional analyses of rRNA mutations inE. coli and most other organisms have been complicated by the presenceof multiple rRNA genes and by the occurrence of dominant lethal rRNAmutations. Because there are seven rRNA operons in E. coli, thephenotypic expression of rRNA mutations may be affected by the relativeamounts of mutant and wild-type ribosomes in the cell. Thus, detectionof mutant phenotypes can be hindered by the presence of wild-typeribosomes. A variety of approaches have been designed to circumventthese problems.

One common approach uses cloned copies of a wild-type rRNA operon(Brosius, J., et al. (1981) Plasmid 6: 112-118; Sigmund, C. D. et al.(1982) Proc. Natl. Acad. Sci. U.S.A. 79: 5602-5606). Several groups haveused this system to detect phenotypic differences caused by a high levelof expression of mutant ribosomes. Recently, a strain of E. coli wasconstructed in which the only supply of ribosomal RNA was plasmidencoded (Asai, T., (1999) J. Bacteriol. 181: 3803-3809). This system hasbeen used to study transcriptional regulation of rRNA synthesis, as wellas ribosomal RNA function (Voulgaris, J., et al. (1999) J. Bacteriol.181: 4170-4175; Koosha, H., et al. (2000) RNA. 6: 1166-1173; Sergiev, P.V., et al. (2000) J. Mol. Biol. 299: 379-389; O'Connor, M. et al. (2001)Nucl. Acids Res. 29: 1420-1425; O'Connor, M., et al. (2001) Nucl. AcidsRes. 29: 710-715; Vila-Sanjurjo, A. et al. (2001) J. Mol. Biol. 308:457-463); Morosyuk S. V., et al. (2000) J. Mol. Biol. 300 (1):113-126;Morosyuk S. V., et al. (2001) J. Mol. Biol. 307 (1):197-210; andMorosyuk S. V., et al. (2001) J. Mol. Biol. 307 (1):211-228. Hui et al.showed that mRNA could be directed to a specific subset ofplasmid-encoded ribosomes by altering the message binding site (MBS) ofthe ribosome while at the same time altering the ribosome binding site(RBS) of an mRNA (Hui, A., et al. (1987) Methods Enzymol. 153: 432-452).

Although each of the above methods has contributed significantly to theunderstanding of rRNA function, progress in this field has been hamperedboth by the complexity of translation and by difficulty in applyingstandard genetic selection techniques to these systems.

Resistance to antibiotics, a matter of growing concern, is caused partlyby antibiotic overuse. According to a study published by the Journal ofthe American Medical Association in 2001, between 1989 to 1999 Americanadults made some 6.7 million visits a year to the doctor for sorethroat. In 73% of those visits, the study found, the patient was treatedwith antibiotics, though only 5%-17% of sore throats are caused bybacterial infections, the only kind that respond to antibiotics.Macrolide antibiotics in particular are becoming extremely popular fortreatment of upper respiratory infections, in part because of theirtypically short, convenient course of treatment. Research has linkedsuch vast use to a rise in resistant bacteria and the recent developmentof multiple drug resistance has underscored the need for antibioticswhich are highly specific and refractory to the development of drugresistance.

Microorganisms can be resistant to antibiotics by four mechanisms.First, resistance can occur by reducing the amount of antibiotic thataccumulates in the cell. Cells can accomplish this by either reducingthe uptake of the antibiotic into the cell or by pumping the antibioticout of the cell. Uptake mediated resistance often occurs, because aparticular organism does not have the antibiotic transport protein onthe cell surface or occasionally when the constituents of the membraneare mutated in a way that interferes with transport of the antibioticinto a cell. Uptake mediated resistance is only possible in instanceswhere the drug gains entry through a nonessential transport molecule.Efflux mechanisms of antibiotic resistance occur via transporterproteins. These can be highly specific transporters that transport aparticular antibiotic, such as tetracycline, out of the cell or they canbe more general transporters that transport groups of molecules withsimilar characteristics out of the cell. The most notorious example of anonspecific transporter is the multi drug resistance transporter (MDR).

Inactivating the antibiotic is another mechanism by which microorganismscan become resistant to antibiotics. Antibiotic inactivation isaccomplished when an enzyme in the cell chemically alters the antibioticso that it no longer binds to its intended target. These enzymes areusually very specific and have evolved over millions of years, alongwith the antibiotics that they inactivate. Examples of antibiotics thatare enzymatically inactivated are penicillin, chloramphenicol, andkanamycin.

Resistance can also occur by modifying or overproducing the target site.The target molecule of the antibiotic is either mutated or chemicallymodified so that it no long binds the antibiotic. This is possible onlyif modification of the target does not interfere with normal cellularfunctions. Target site overproduction is less common but can alsoproduce cells that are resistant to antibiotics.

Lastly, target bypass is a mechanism by which microorganisms can becomeresistant to antibiotics. In bypass mechanisms, two metabolic pathwaysor targets exist in the cell and one is not sensitive to the antibiotic.Treatment with the antibiotic selects cells with more reliance on thesecond, antibiotic-resistant pathway.

Among these mechanisms, the greatest concern for new antibioticdevelopment is target site modification. Enzymatic inactivation andspecific transport mechanisms require the existence of a substratespecific enzyme to inactivate or transport the antibiotic out of thecell. Enzymes have evolved over millions of years in response tonaturally occurring antibiotics. Since microorganisms cannotspontaneously generate new enzymes, these mechanisms are unlikely topose a significant threat to the development of new syntheticantibiotics. Target bypass only occurs in cells where redundantmetabolic pathways exist. As understanding of the MDR transportersincreases, it is increasingly possible to develop drugs that are nottransported out of the cell by them. Thus, target site modificationposes the greatest risk for the development of antibiotic resistance fornew classes of antibiotic and this is particularly true for thoseantibiotics that target ribosomes. The only new class of antibiotics inthirty-five years, the oxazolidinones, is a recent example of anantibiotic that has been compromised because of target sitemodification. Resistant strains containing a single mutation in rRNAdeveloped within seven months of its use in the clinical settings.

BRIEF SUMMARY OF THE INVENTION

The “instant evolution” system was initially developed in E. coli,primarily because of the ease with which this organism can begenetically manipulated. [WO 2004/003511.] Because many of thefunctionally important regions of rRNA are conserved among bacteria,drug leads developed against conserved targets in the E. coli system mayproduce broad-spectrum anti-infectives. However, in order to develop asystem to produce narrow-spectrum anti-infectives, provided herein aremethods and compositions for screening Pseudomonas aeruginosa 16S rRNAin E. coli cells. In certain embodiments, a plasmid comprising the 16SrRNA gene from Pseudomonas aeruginosa, mutated to replace the naturalhelix 9 region with the corresponding region of the E. coli rRNA, isprovided to form functional ribosomes in E. coli host cells. In otherembodiments, a plasmid, comprising the unmutated 16S rRNA fromPseudomonas aeruginosa, along with a plasmid containing the Pseudomonasaeruginosa S20 protein, is provided which can yield functional ribosomesin E. coli cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the plasmid construct Pa 16S pRNA228 in the bottom panel,wherein the E. coli 16S is replaced with the P. aeruginosa between theE. coli Bcll and EstEII sites. The locations of specific sites in Pa 16SpRNA228 are shown in the upper panel as follows: the 16S rRNA E. colirrnB operon corresponds to nucleic acids 1-14 & 1499-1536; the 16S rRNAP. aeruginosa rrnC operon corresponds to nucleic acids 15-1498; the 16SMBS (message binding sequence) GGGAU corresponds to nucleic acids1530-1534; the 16S-23S spacer region corresponds to nucleic acids1537-1976; the 23S rRNA of E. coli rrnB operon corresponds to nucleicacids 1977-4880; the 23S-5S spacer region corresponds to nucleic acids4881-4972; the 5S rRNA of E. coli rrnB operon corresponds to nucleicacids 4973-5092; the terminator T1 of E. coli rrnB operon corresponds tonucleic acids 5096-5139; the terminator T2 of E. coli rrnB operoncorresponds to nucleic acids 5271-5299; the bla (β-lactamase; ampicillinresistance) corresponds to nucleic acids 6462-7319; the replicationorigin corresponds to nucleic acids 7482-8096; the rop (Rop protein)corresponds to nucleic acids 8509-8700; the GFP corresponds to nucleicacids 9113-10088; the GFP RBS (ribosome binding sequence) AUCCCcorresponds to nucleic acids 10096-10100; the trp^(c) promotercorresponds to nucleic acids 10117-10157; the trp^(c) promotercorresponds to nucleic acids 10632-10672; the CAT RBS AUCCC correspondsto nucleic acids 10689-10693; the cam (chloramphenicolacetyltransferase: CAT) corresponds to nucleic acids 10701-11630; thelacY¹ promoter corresponds to nucleic acids 11670-11747; the /ac/q (lacrepressor) corresponds to nucleic acids 11748-12830; and the lacUV5promoter corresponds to nucleic acids 12873-12914.

FIG. 2A-B depict the plasmid construct Pa 16S Ec H9 pRNA228. In FIG. 2B,the plasmid map is shown wherein the E. coli 16S is replaced with the P.aeruginosa between the E. coli Bcll and EstEII sites and the P.aeruginosa helix 9 (H9) sequence is replaced with the E. coli H9sequence. FIG. 2A shows that the plasmid is made up of E. coli rRNAsequence at positions 1-14, 176-188 and 1499-1536; and P. aeruginosarRNA sequence at positions 15-175 and 189-1489. The E. coli H9 sequenceis AUAACGUCGCAAGACCAAA (SEQ ID NO: 1) and the P. aeruginosa H9 sequenceis

(SEQ ID NO: 2) AUACGUCCUGAGGGAGAAA.10 mutations were made and they are denoted with an underline in the P.aeruginosa H9 sequence shown above.

FIG. 3 depicts a pKan5-T1T2 vector in the bottom panel wherein the E.coli terminators from pRNA228 were moved (via PCR) into the multicloningsite of pKANS using restriction enzymes XbaI and EcoRI. The locations ofspecific sites in pKan5-T1T2 are shown in the upper panel as follows:the replication origin corresponds to nucleic acids 723-1268; aph(3′)-la (kanamycin resistance) corresponds to nucleic acids 1862-2677;araC corresponds to nucleic acids 2950-3828; Pc (the araC promoter)corresponds to nucleic acids 3979-4005; the pBAD promoter corresponds tonucleic acids 4104-4131; the terminator T1 of E. coli rrnB operoncorresponds to nucleic acids 4163-4206; and the terminator T2 of E. colirrnB operon corresponds to nucleic acids 4338-4366.

FIG. 4 depicts a pKan5 vector in the bottom panel wherein the the E.coli terminators from pRNA228 were moved (via PCR) into the multicloningsite of pKan5 using restriction enzymes XbaI and EcoRI; and the P.aeruginosa S20 protein was also cloned into the vector using enzymesNotI and XbaI (denoted herein as “pKanPa-S20”). The locations ofspecific sites in pKan5Pa-S20 are shown in the upper panel as follows:the replication origin corresponds to nucleic acids 723-1268; aph (3)-la(kanamycin resistance) corresponds to nucleic acids 1862-2677; araCcorresponds to nucleic acids 2950-3828; Pc (the araC promoter)corresponds to nucleic acids 3979-4005; the pBAD promoter corresponds tonucleic acids 4104-4131; the Shine-Dalgarno sequence (GAGGA) correspondsto nucleic acids 4168-4172; P. aeruginosa S20 corresponds to 4181-4453;the terminator T1 of E. coli rrnB operon corresponds to nucleic acids4463-4506; and the terminator T2 of E. coli rrnB operon corresponds tonucleic acids 4638-4666.

FIG. 5 depicts the results of green fluorescent protein (GFP) assays forP. aeruginosa constructs.

FIGS. 6 a-f depict the sequence of the plasmid depicted in FIG. 1 (SEQID NO: 3). FIGS. 7 a-f depict the sequence of the plasmid depicted inFIG. 2 (SEQ ID NO: 4). FIGS. 8 a-b depict the sequence of the plasmiddepicted in FIG. 3 (SEQ ID NO: 5).

FIGS. 9 a-b depict the sequence of the plasmid depicted in FIG. 4 (SEQID NO: 6).

FIG. 10 depicts novel mutant anti-Shine-Dalgarno (ASD) sequences (SEQ IDNOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32) and novelmutant Shine-Dalgarno (SD) sequences (SEQ ID NOs: 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, and 31) of the present invention. FIG. 10 alsoshows a sequence analysis of chloramphenicol resistant isolates. Themutated nucleotides are underlined and potential duplex formations areboxed. CAT activity was measured twice for each culture and the unit isCPM/0.1 μL of culture/OD600. Induction was measured by dividing CATactivity in induced cells with CAT activity in uninduced cells. A −1indicates no induction, while a +1 indicates induction with 1 mM IPTG.

FIG. 11 depicts novel mutant ASD sequences (SEQ ID NOs: 34, 36, 38, 40,42, 44, 46 and 48)and novel mutant SD sequences (SEQ ID NOs: 33, 35, 37,39, 41, 43, 45 and 47), of the present invention. FIG. 11 also shows asequence analysis of CAT mRNA mutants (SEQ ID NOs: 34, 36, 38, 40, 42,44, 46 and 48) . Potential duplex formations are boxed and the mutatednucleotides are underlined. The start codon (AUG) is in bold. −1 in thetable indicates no induction, whereas +1 indicates induction with 1 mMIPTG.

FIGS. 12 a-b depict novel nucleic acid mutant ASD sequences and novelcomplementary mutant SD sequences. FIG. 12A-1 depicts novel mutant ASDsequences SEQ ID NOs: 50, 52, 54, 56 and 58 and novel mutant SDsequences SEQ ID NOs: 49, 51, 53, 55, and 57; FIG. 12A-2 depicts novelmutant ASD sequences SEQ ID NOs: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78and 80 and novel mutant SD sequences SEQ ID NOs: 59, 61, 63, 65, 67, 69,71, 73, 75, 77 and 79, FIG. 12A-3 depicts novel mutant ASD sequences 82,84, 86, 88, 90, 92, 94, 96, 98 and 100 and novel mutant SD sequences SEQID NOs: 81, 83, 85, 87, 89, 91, 93, 95, 97 and 99; FIG. 12B-1 depictsnovel mutant ASD sequences SEQ ID NOs: 102, 104, and 106 and novelmutant SD sequences SEQ ID NOs: 101, 103 and 105; FIG. 12B-2 depictsnovel ASD sequences SEQ ID NOs: 108, 110, 112, 114, 116, 118, 120, 122,124, 126, and 128 and novel SD sequences SEQ ID NOs: 107, 109, 111, 113,115, 117, 119, 121, 123, 125, and 127, and FIG. 12B-3 depicts novel ASDsequences SEQ ID NOs: 130, 132, 134, 136, 138, 140, 142, 144, 146 and148 and novel SD sequences SEQ ID NOs: 129, 131, 133, 135, 137, 139,141, 143, 145 and 147.

FIG. 13 a-i depict P. aeruginosa 16S rRNA (SEQ ID NO: 149), with theshown nucleotide differences between E. coli (SEQ ID NO: 150) and P.aeruginosa 16S rRNA (SEQ ID NO: 149). The P. aeruginosa 16S rRNA isshown in full, with the E. coli sequence differences shown in circles.

FIG. 14 depicts a tabulation of 16s rRNA hybrid P. aeruginosa and E.coli sequences and the resulting measured GFP percentage. (GFP 100% wasset for E. coli sequence with no P. aeruginosa sequence.) The last entryin the table demonstrates the significance of nucleotides 176-188; thisis shown pictorially in FIG. 15.

FIG. 15 a-h depict a mutant P. aeruginosa 16S rRNA wherein the helix 9(H9) nucleotides, numbered 176-188 (boxed in the lower left) have beenreplaced with the corresponding E. coli H9 nucleotides (SEQ ID NO: 151).

FIG. 16 depicts the ribosomal protein S20 binding site on P. aeruginosa16S rRNA (SEQ ID NO: 152).

FIG. 17 depicts an S20 protein alignment of E. coli (SEQ ID NO: 154)compared with P. aeruginosa (SEQ ID NO: 153) and Thermus species (SEQ IDNO: 155).

FIG. 18 depicts a schematic of the addition of a plasmid containing theS20 gene into transformed in cells containing the 16S rRNA of E. coliand P. aeruginosa, and the measurement of resulting activity by GFP.

In addition, FIGS. 1-26 of WO 2004/003511 (some of which may also befound in Lee, K., et al. Genetic Approaches to Studying ProteinSynthesis: Effects of Mutations at Pseudouridine 516 and A535 inEscherichia coli 16S rRNA. Symposium: Translational Control: AMechanistic Perspective at the Experimental Biology 2001 Meeting (2001);and Lee, K., et al., J. Mol. Biol. 269: 732-743 (1997)) are expresslyincorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

Overview. The “instant evolution” system (as described in WO2004/003511; herein incorporated by reference in its entirety) wasinitially developed in E. coli, primarily because of the ease with whichthis organism can be genetically manipulated. Because many of thefunctionally important regions of rRNA are conserved among bacteria,drug leads developed against conserved targets in the E. coli system mayproduce broad-spectrum anti-infectives.

Recently, however, emphasis has been placed on the development of narrowspectrum antimicrobials because of the reduced likeliness of adverseeffects and because resistance to narrow-spectrum antibiotics is lesslikely to occur. Several rRNA genes from human pathogens were thereforetested to see whether their genes could be expressed in E. coli.

Compositions and Methods of the Invention. In one embodiment,compositions and methods are provided to identify functional mutant P.aeruginosa ribosomes suitable as drug targets. The compositions andmethods of the invention allow for the expression of P. aeruginosaribosomes in E. coli. The compositions and methods further allow for theisolation and analysis of mutations that would normally be lethal andallow direct selection of rRNA mutants with predetermined levels ofribosome function. The compositions and methods of the present inventionmay additionally be used to identify antibiotics to treat generallyand/or selectively, human pathogens such as P. aeruginosa.

According to one embodiment of the invention, a functional genomicsdatabase for 16S rRNA genes of a variety of species is generated. Inparticular, the rRNA gene is randomly mutated using a generalizedmutational strategy. A host E. coli cell is then transformed with amutagenized plasmid of the invention comprising: an P. aeruginosa rRNAgene having a mutant ASD sequence, the mutated P. aeruginosa rRNA gene,and a genetically engineered gene which encodes a selectable markerhaving a mutant SD sequence. In certain embodiments, said P. aeruginosaDNA is further modified so as to replace helix 9 with the correspondingE. coli helix (FIG. 15); in other embodiments the host cell is furthertransformed with a plasmid encoding the P. aeruginosa S20 gene. Theselectable marker gene, such as CAT, may be used to select mutants thatare functional, e.g., by plating the transformed cells onto growthmedium containing chloramphenicol. The mutant rRNA genes contained ineach plasmid DNA of the individual clones from each colony are selectedand characterized. The function of each of the mutant rRNA genes isassessed by measuring the amount of an additional selectable markergene, such as GFP, produced by each clone upon induction of the rRNAoperon. A functional genomics database may thus be assembled, whichcontains the sequence and functional data of the functional mutant rRNAgenes. In particular, functionally important regions of the rRNA genethat will serve as drug targets are identified by comparing thesequences of the functional genomics database and correlating thesequence with the amount of GFP protein produced.

In another embodiment, the nucleotides in the functionally importanttarget regions identified in the above methods may be simultaneouslyrandomly mutated, e.g., by using standard methods of molecularmutagenesis, and cloned into a plasmid of the invention to form aplasmid pool containing random mutations at each of the nucleotidepositions in the target region. The resulting pool of plasmidscontaining random mutations is then used to transform cells, e.g., E.coli cells, and form a library of clones, each of which contains aunique combination of mutations in the target region. The library ofmutant clones are grown in the presence of IPTG to induce production ofthe mutant rRNA genes and a selectable marker is used, such as CAT, toselect clones of rRNA mutants containing nucleotide combinations of thetarget region that produce functional ribosomes. The rRNA genesproducing functional ribosomes are sequenced and may be incorporatedinto a database.

In yet another embodiment, a series of oligonucleotides may besynthesized that contain the functionally-important nucleotides andnucleotide motifs within the target region and may be used tosequentially screen compounds and compound libraries to identifycompounds that recognize (bind to) the functionally important sequencesand motifs. The compounds that bind to all of the oligonucleotides arethen counter-screened against oligonucleotides and/or other RNAcontaining molecules to identify drug candidates. Drug candidatesselected by the methods of the present invention are thus capable ofrecognizing all of the functional variants of the target sequence, i.e.,the target cannot be mutated in a way that the drug cannot bind, withoutcausing loss of function to the ribosome.

In still another embodiment, after the first stage mutagenesis of theentire rRNA is performed using techniques known in the art, e.g.,error-prone PCR mutagenesis, the mutants are analyzed to identifyregions within the rRNA that are important for function. These regionsare then sorted based on their phylogenetic conservation, as describedherein, and are then used for further mutagenesis.

Ribosomal RNA sequences from each species are different and the moreclosely related two species are, the more their rRNAs are alike. Forinstance, humans and monkeys have very similar rRNA sequences, buthumans and bacteria have very different rRNA sequences. Thesedifferences may be utilized for the development of very specific drugswith a narrow spectrum of action and also for the development ofbroad-spectrum drugs that inhibit large groups of organisms that areonly distantly related, such as all bacteria.

In another embodiment, the functionally important regions identifiedabove are divided into groups based upon whether or not they occur inclosely related groups of organisms. For instance, some regions of rRNAare found in all bacteria but not in other organisms. Other areas ofrRNA are found only in closely related groups of bacteria, such as allof the members of a particular species, e.g., members of the genusMycobacterium or Streptococcus.

In a further embodiment, the regions found in very large groups oforganisms, e.g., all bacteria or all fungi, are used to developbroad-spectrum antibiotics that may be used to treat infections from alarge number of organisms within that group. The methods of the presentinvention may be performed on these regions and functional mutantribosomes identified. These functional mutant ribosomes may be screened,for example, with compound libraries.

In yet another embodiment, regions that are located only in relativelysmall groups of organisms, such as all members of the genusStreptococcus or all members of the genus Mycobacterium, may be used todesign narrow spectrum antibiotics that will only inhibit the growth oforganisms that fall within these smaller groups. The methods of thepresent invention may be performed on these regions and functionalmutant ribosomes identified. These functional mutant ribosomes will bescreened, e.g., compound libraries.

The invention provides novel plasmid constructs, e.g. those depicted inFIGS. 1 to 4. In addition, the novel plasmid constructs of the presentinvention employ novel mutant anti-Shrine-Delgano (ASD) and mutantShine-Delgano (SD) sequences set forth in FIGS. 10, 11 and 12. Themutant ASD and mutant SD sequences may be used as mutually compatiblepairs (see FIGS. 10, 11 and 12). It will be appreciated that themutually compatible pairs of mutant ASD and SD sequences interact aspairs in the form of RNA, to permit translation of only the mRNAscontaining the altered SD sequence.

Definitions. For convenience, certain terms employed in thespecification, examples, and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a living cell substantiallyonly when an inducer which corresponds to the promoter is present in thecell.

As used herein, the term “mutation” includes an alteration in thenucleotide sequence of a given gene or regulatory sequence from thenaturally occurring or normal nucleotide sequence. A mutation may be asingle nucleotide alteration (e.g., deletion, insertion, substitution,including a point mutation), or a deletion, insertion, or substitutionof a number of nucleotides.

By the term “selectable marker” is meant a gene whose expression allowsone to identify functional mutant ribosomes. Useful selectable markersare well known in the art and include, for example, antibiotic andherbicide resistance genes, gas concentrations, nutrients, and wasteproducts. Specific examples of such genes are disclosed in Weising etal. Weising, K, et al., (1988) Ann Rev of Genetics 22:421-478; thecontents of which are incorporated by reference. Examples of selectablemarker genes are genes encoding a protein such as G418 and hygromycinwhich confer resistance to certain drugs, for example, β-galactosidase,chloramphenicol acetyltransferase, or firefly luciferase. Transcriptionof the selectable marker gene is monitored by changes in theconcentration of the selectable marker protein such as β-galactosidase,chloramphenicol acetyltransferase, or firefly luciferase. If theselectable marker gene encodes a protein conferring antibioticresistance such as neomycin resistance transformant cells can beselected with G418. Cells that have incorporated the selectable markergene will survive, while the other cells die. This makes it possible tovisualize and assay for expression of recombinant expression vectors ofthe invention and in particular to determine the effect of a mutation onexpression and phenotype. It will be appreciated that selectable markerscan be introduced on a separate vector from the nucleic acid ofinterest.

The term ‘Gram-positive bacteria’ is an art recognized term for bacteriacharacterized by having as part of their cell wall structurepeptidoglycan as well as polysaccharides and/or teichoic acids and arecharacterized by their blue-violet color reaction in the Gram-stainingprocedure. Representative Gram-positive bacteria include: Actinomycesspp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum,Clostridium perfringens, Clostridium spp., Clostridium tetani,Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcusfaecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae,Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum,Leuconostoc spp., Mycobacterium abcessus, Mycobacterium avium complex,Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacteriumhaemophilium, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacteriumsmegmatis, Mycobacterium terrae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,Peptostreptococcus spp., Proprionibacterium spp., Staphylococcus aureus,Staphylococcus auricularis, Staphylococcus capitis, Staphylococcuscohnii, Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdanensis, Staphylococcussaccharolyticus, Staphylococcus saprophyticus, Staphylococcusschleiferi, Staphylococcus similans, Staphylococcus warneri,Staphylococcus xylosus, Streptococcus agalactiae (group Bstreptococcus), Streptococcus anginosus, Streptococcus bovis,Streptococcus canis, Streptococcus equi, Streptococcus milleri,Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae,Streptococcus pyogenes (group A streptococcus), Streptococcussalivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” is an art recognized term for bacteriacharacterized by the presence of a double membrane surrounding eachbacterial cell. Representative Gram-negative bacteria includeAcinetobacter calcoaceticus, Actinobacillus actinomycetemcomitans,Aeromonas hydrophila, Alcaligenes xylosoxidans, Bacteroides, Bacteroidesfragilis, Bartonella bacilliformis, Bordetella spp., Borreliaburgdorferi, Branhamella catarrhalis, Brucella spp., Campylobacter spp.,Chalmydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis,Chromobacterium violaceum, Citrobacter spp., Eikenella corrodens,Enterobacter aerogenes, Escherichia coli, Flavobacteriummeningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilusspp., Helicobacter pylori, Klebsiella spp., Legionella spp., Leptospiraspp., Moraxella catarrhalis, Morganella morganii, Mycoplasma pneumoniae,Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida,Plesiomonas shigelloides, Prevotella spp., Proteus spp., Providenciarettgeri, Pseudomonas aeruginosa, Pseudomonas spp., Rickettsiaprowazekii, Rickettsia rickettsii, Rochalimaea spp., Salmonella spp.,Salmonella typhi, Serratia marcescens, Shigella spp., Treponemacarateum, Treponema pallidum, Treponema pallidum endemicum, Treponemapertenue, Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, Yersinia pestis.

Isolated Nucleic Acid Molecules. As used herein, the term “nucleic acidmolecule” is intended to include DNA molecules (e.g., cDNA or genomicDNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNAgenerated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture medium,when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence set forth in FIGS. 10, 11, and12, or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Usingall or portion of the nucleic acid sequence set forth in FIGS. 10, 11,and 12 as a hybridization probe, the nucleic acid molecules of thepresent invention can be isolated using standard hybridization andcloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F.,and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of thesequence set forth in FIGS. 10, 11, and 12, can be isolated by thepolymerase chain reaction (PCR) using synthetic oligonucleotide primersdesigned based upon the sequence set forth in FIGS. 10, 11, and 12.

A nucleic acid of the invention can be amplified using cDNA, mRNA or,alternatively, genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to the nucleotide sequences of the present invention canbe prepared by standard synthetic techniques, e.g., using an automatedDNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence set forth in FIGS. 10, 11, and 12, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in FIGS. 10, 11, and 12is one which is sufficiently complementary to the nucleotide sequenceshown in FIGS. 10, 11, and 12, such that it can hybridize to thenucleotide sequence shown in FIGS. 10, 11, and 12, respectively, therebyforming a stable duplex.

“Homology” or alternatively “identity” refers to sequence similaritybetween two nucleic acid molecules. Homology may be determined bycomparing a position in each sequence, which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base, then the molecules are homologous at that position. Adegree of homology between sequences is a function of the number ofmatching or homologous positions shared by the sequences.

The term “percent identical” refers to sequence identity between twonucleotide sequences. Identity may be determined by comparing a positionin each sequence, which may be aligned for purposes of comparison. Whenan equivalent position in the compared sequences is occupied by the samebase or amino acid, then the molecules are identical at that position;when the equivalent site is occupied by the same or a similar base(e.g., similar in steric and/or electronic nature), then the moleculesmay be referred to as homologous (similar) at that position. Expressionas a percentage of homology, similarity, or identity refers to afunction of the number of identical or similar amino acids at positionsshared by the compared sequences. Various alignment algorithms and/orprograms may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLASTare available as a part of the GCG sequence analysis package (Universityof Wisconsin, Madison, Wis.), and may be used with, e.g., defaultsettings. ENTREZ is available through the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md. In one embodiment, the percentidentity of two sequences may be determined by the GCG program with agap weight of 1, e.g., each nucleic acid gap is weighted as if it were asingle nucleotide mismatch between the two sequences. Other techniquesfor alignment are described in Methods in Enzymology, vol. 266: ComputerMethods for Macromolecular Sequence Analysis (1996), ed. Doolittle,Academic Press, Inc., a division of Harcourt Brace & Co., San Diego,Calif., USA. Preferably, an alignment program that permits gaps in thesequence is utilized to align the sequences. The Smith-Waterman is onetype of algorithm that permits gaps in sequence alignments. See Meth.Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needlemanand Wunsch alignment method may be utilized to align sequences. Analternative search strategy uses MPSRCH software, which runs on a MASPARcomputer. MPSRCH uses a Smith-Waterman algorithm to score sequences on amassively parallel computer. This approach improves the ability to pickup distantly related matches, and is especially tolerant of small gapsand nucleotide sequence errors. Nucleic acid-encoded amino acidsequences may be used to search both protein and DNA databases.Databases with individual sequences are described in Methods inEnzymology, ed. Doolittle, supra. Databases include Genbank, EMBL, andDNA Database of Japan (DDBJ).

The terms “polynucleotide”, and “nucleic acid” are used interchangeablyto refer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof Thefollowing are non-limiting examples of polynucleotides: coding ornon-coding regions of a gene or gene fragment, loci (locus) defined fromlinkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA,ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and nucleotide analogs. If present, modifications to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.The term “recombinant” polynucleotide means a polynucleotide of genomic,cDNA, semi-synthetic, or synthetic origin, which either does not occurin nature or is linked to another polynucleotide in a non-naturalarrangement. An “oligonucleotide” refers to a single strandedpolynucleotide having less than about 100 nucleotides, less than about,e.g., 75, 50, 25, or 10 nucleotides.

The term “specifically hybridizes” refers to detectable and specificnucleic acid binding. Polynucleotides, oligonucleotides and nucleicacids of the invention selectively hybridize to nucleic acid strandsunder hybridization and wash conditions that minimize appreciableamounts of detectable binding to nonspecific nucleic acids. Stringentconditions may be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, andnucleic acids of the invention and a nucleic acid sequence of interestwill be at least 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, 99%,or more. In certain instances, hybridization and washing conditions areperformed under stringent conditions according to conventionalhybridization procedures and as described further herein.

The terms “stringent conditions” or “stringent hybridization conditions”refer to conditions, which promote specific hybridization between twocomplementary polynucleotide strands so as to form a duplex. Stringentconditions may be selected to be about 5° C. lower than the thermalmelting point (Tm) for a given polynucleotide duplex at a defined ionicstrength and pH. The length of the complementary polynucleotide strandsand their GC content will determine the Tm of the duplex, and thus thehybridization conditions necessary for obtaining a desired specificityof hybridization. The Tm is the temperature (under defined ionicstrength and pH) at which 50% of a polynucleotide sequence hybridizes toa perfectly matched complementary strand. In certain cases it may bedesirable to increase the stringency of the hybridization conditions tobe about equal to the Tm for a particular duplex.

A variety of techniques for estimating the Tm are available. Typically,G-C base pairs in a duplex are estimated to contribute about 3° C. tothe Tm, while A-T base pairs are estimated to contribute about 2° C., upto a theoretical maximum of about 80-100° C. However, more sophisticatedmodels of Tm are available in which G-C stacking interactions, solventeffects, the desired assay temperature and the like are taken intoaccount. For example, probes can be designed to have a dissociationtemperature (Td) of approximately 60° C., using the formula:Td=(((((3×#GC)+(2×#AT))×37)−562)/#bp)−5; where #GC, #AT, and #bp are thenumber of guanine-cytosine base pairs, the number of adenine-thyminebase pairs, and the number of total base pairs, respectively, involvedin the formation of the duplex.

Hybridization may be carried out in SxSSC, 4xSSC, 3xSSC, 2xSSC, 1xSSC or0.2xSSC for at least about 1 hour, 2 hours, 5 hours, 12 hours, or 24hours. The temperature of the hybridization may be increased to adjustthe stringency of the reaction, for example, from about 25° C. (roomtemperature), to about 45° C., 50° C., 55° C., 60° C., or 65° C. Thehybridization reaction may also include another agent affecting thestringency, for example, hybridization conducted in the presence of 50%formamide increases the stringency of hybridization at a definedtemperature.

The hybridization reaction may be followed by a single wash step, or twoor more wash steps, which may be at the same or a different salinity andtemperature. For example, the temperature of the wash may be increasedto adjust the stringency from about 25° C. (room temperature), to about45° C., 50° C., 55° C., 60° C., 65° C., or higher. The wash step may beconducted in the presence of a detergent, e.g., 0.1 or 0.2% SDS. Forexample, hybridization may be followed by two wash steps at 65° C. eachfor about 20 minutes in 2xSSC, 0.1% SDS, and optionally two additionalwash steps at 65° C. each for about 20 minutes in 0.2xSSC, 0.1% SDS.

Exemplary stringent hybridization conditions include overnighthybridization at 65° C. in a solution comprising, or consisting of, 50%formamide, 10xDenhardt (0.2% Ficoll, 0.2% Polyvinylpyrrolidone, 0.2%bovine serum albumin) and 200 μg/ml of denatured carrier DNA, e.g.,sheared salmon sperm DNA, followed by two wash steps at 65° C. each forabout 20 minutes in 2xSSC, 0.1% SDS, and two wash steps at 65° C. eachfor about 20 minutes in 0.2xSSC, 0.1% SDS.

Hybridization may consist of hybridizing two nucleic acids in solution,or a nucleic acid in solution to a nucleic acid attached to a solidsupport, e.g., a filter. When one nucleic acid is on a solid support, aprehybridization step may be conducted prior to hybridization.Prehybridization may be carried out for at least about 1 hour, 3 hoursor 10 hours in the same solution and at the same temperature as thehybridization solution (without the complementary polynucleotidestrand).

Appropriate stringency conditions are known to those skilled in the artor may be determined experimentally by the skilled artisan. See, forexample, Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-12.3.6; Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, N.Y; S. Agrawal (ed.)Methods in Molecular Biology, volume 20; Tijssen (1993) LaboratoryTechniques in biochemistry and molecular biology-hybridization withnucleic acid probes, e.g., part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays”, Elsevier,New York; and Tibanyenda, N. et al., Eur. J. Biochem. 139:19 (1984) andEbel, S. et al., Biochem. 31:12083 (1992).

The term “substantially homologous” when used in connection with anucleic acid or amino acid sequences, refers to sequences which aresubstantially identical to or similar in sequence with each other,giving rise to a homology of conformation and thus to retention, to auseful degree, of one or more biological (including immunological)activities. The term is not intended to imply a common evolution of thesequences.

Recombinant Expression Vectors and Host Cells. Another aspect of theinvention pertains to vectors, preferably expression vectors, containinga nucleic acid molecule of the present invention (or a portion thereof).As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRITS (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al. (1988) Gene 69:301-315) and pET lld (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET lldvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gnl). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident prophage harboring a T7 gnl gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al. (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector may be a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSecl (Baldari et al. (1987) Embo J. 6:229-234), pMFa (Kurjanand Herskowitz (1982) Cell 30:933-943), pJRY88 (Schultz et al. (1987)Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (Invitrogen Corp, San Diego, Calif.).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY, 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235-275), particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example by the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379).

Another aspect of the invention pertains to host cells into which a thenucleic acid molecule of the invention is introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

A host cell can be any prokaryotic or eukaryotic cell. Other suitablehost cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

Making P. aeruginosa 16S rRNA active in E. coli. As already mentionedherein, a plasmid comprising the 16S rRNA gene from P. aeruginosa,mutated to replace the natural helix 9 region with the correspondingregion of the E. coli rRNA, is provided to form functional mutantribosomes. In addition, in certain embodiments, a plasmid comprising thePseudomonas aeruginosa S20 protein is provided, thereby yieldingfunctional ribosomes in E. coli cells.

There are many different ribosomal proteins. The 30S ribosomal proteinsare designated S1-S21. It is known that S4, S7, S8, S15, S17 and S20bind independently to 16S rRNA. Binding of these primary bindingproteins folds the rRNA and allows binding of the secondary bindingproteins (S5, S6, S9, S12, S13, S16, S18, and S19). These proteins alsofold the ribosome to allow the addition of S2, S3, S10, S11, S14 and S21(which are known as tertiary binding proteins).

The ribosomal protein S20 binding site on P. aeriginosa are shown inFIG. 16. 16S rRNA Protein alignments for E. coli and P. aeruginosa areshown in FIG. 17. As shown therein, S20′s pH2 and pH3 interact with H9of the 16S rRNA. It is hypothesized that charge differences between E.coli and P. aeruginosa S20 composition may play a role in binding of theprotein to the rRNA.

The expression of P. aeruginosa S20 can be made in the presence of E.coli and P. aeruginosa 16S RNA. One begins by obtaining chromosomal DNAfrom P. aeruginosa. Then the S20 gene is PCRed. Next, this gene iscloned into an expression vector. Finally, the plasmid containing theS20 gene is transformed in cells (e.g. DH5 cells) containing the 16SrRNA of E. coli and P. aeruginosa, and the activity is measured. Aschematic of this process is shown in FIG. 18. GFP analysis of P.aeruginosa (46% activity of ribosomes without P. aeruginosa S20 and96.4% activity of ribosomes with P. aeruginosa S20) confirms that P.aeruginosa 16S rRNA is complemented by P. aeruginosa S20. When thevector only was assayed (i.e. no S20) no increase in ribosomal activitywas observed. P. aeruginosa 16S rRNAs may therefore be made active in E.coli by replacing helix 9 of P. aeruginosa 16S rRNA with thecorresponding sequence from E. coli or by adding P. aeruginosa S20 tocells that are making the P. aeruginosa 16S rRNA. Both constructs can beused to develop antibiotics that target P. aeruginosa 16S rRNA.

Making other pathogen's 16S rRNA active in E. coli. Given P. aeruginosa16S rRNA can be fully expressed in E. coli, it is likely that the rRNAsfrom other pathogens (e.g. Gram-positive or Gram-negative bacteria) canalso be expressed in E. coli. This will facilitate the identification ofnew antibiotic targets, and therefore new classes of antibiotics whichare less susceptible to the development of resistance. The inventivesystem described herein can be used to identify antibiotic leads thatdisrupt the interaction between critical nucleotides of pathogenic 16SrRNA and the critical amino acids on ribosomal proteins (such ascritical nucleotides on P. aeruginosa 16S rRNA and P. aeruginosa S20).An antibiotic developed in this manner with specifically target thepathogen used, and its close relatives, without effecting otherbacteria.

Uses and Methods of the Invention. The nucleic acid molecules describedherein may be used in a plasmid construct, e.g. pRNA228, to carry outone or more of the following methods: (1) creation of a functionalgenomics database of the rRNA genes generated by the methods of thepresent invention; (2) mining of the database to identify functionallyimportant regions of the rRNA; (3) identification of functionallyimportant sequences and structural motifs within each target region; (4)screening compounds and compound libraries against a series offunctional variants of the target sequence to identify compounds thatbind to all functional variants of the target sequence; and (5)counterscreening the compounds against nontarget RNAs, such as humanribosomes or ribosomal RNA sequences.

One aspect of the invention relates to a plasmid comprising a firstnucleic acid sequence and a second nucleic acid sequence; wherein saidfirst nucleic acid sequence encodes a Pseudomonas aeruginosa 16S rRNAcomprising a mutant Anti-Shine-Dalgarno sequence and at least oneadditional mutation outside the Anti-Shine-Dalgarno region; and saidsecond sequence encodes a selectable marker having a mutantShine-Dalgarno sequence; wherein said mutant Anti-Shine-Dalgarnospecifically hybridizes to said mutant Shine-Dalgarno sequence.

Another aspect of the invention relates to the plasmid of FIG. 1.

Another aspect of the invention relates to a plasmid comprising a firstnucleic acid sequence and a second nucleic acid sequence; wherein saidfirst nucleic acid sequence encodes a Pseudomonas aeruginosa 16S rRNAcomprising a mutant Anti-Shine-Dalgarno sequence, a mutant helix 9sequence, and at least one additional mutation outside theAnti-Shine-Dalgarno region; and said second sequence encodes aselectable marker having a mutant Shine-Dalgarno sequence; wherein saidmutant Anti-Shine-Dalgarno specifically hybridizes to said mutantShine-Dalgarno sequence.

Another aspect of the invention relates to the plasmid of FIG. 2.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the selectable marker is chosen from thegroup consisting of chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), and both CAT and GFP.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein said mutant helix 9 sequence is SEQ IDNO: 1.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the mutant Anti-Shine-Dalgarno sequenceis selected from the group consisting of the sequences set forth inFIGS. 10, 11 and 12.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the mutant Shine-Dalgarno sequence isselected from the group consisting of the sequences set forth in FIGS.10, 11 and 12.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the mutant Anti-Shine-Dalgarno sequenceand the mutant SD sequence are a complementary pair selected from thegroup consisting of the sequences set forth in FIGS. 10,11 and 12.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the complementary mutant Shine-Dalgarnoand mutant Anti-Shine-Dalgarno pair permits translation by the rRNA ofthe selectable marker.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the selectable marker is CAT.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the selectable marker is GFP.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the DNA sequence encoding the rRNA geneis under the control of an inducible promoter.

Another aspect of the invention relates to a plasmid comprising aPseudomonas aeruginosa S20 gene.

Another aspect of the invention relates to a plasmid of FIG. 3 or 4.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising aph (3′)-la (kanamycinresistance) gene.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising the terminator T1 andterminator T2 of the Escherichia coli rrnA, rrnB, rrnC, rrnD, rrnE, rrnGor rrnH operon; or a T1 terminator from one and a T2 terminator fromanother.

In certain embodiments the present invention relates to theaforementioned plasmid, comprising an operon from a bacteria.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising the terminator T1 andterminator T2 of the Escherichia coli rrnB operon.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising the terminator T1 andterminator T2 of the Escherichia coli rrnC operon.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising the terminator T1 andterminator T2 of an Escherichia coli rrn operon.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising the pBAD promotor and theAraC activator.

In certain embodiments the present invention relates to theaforementioned plasmid, further comprising the Shine-Dalgarno sequenceGAGGA.

In certain embodiments the present invention relates to theaforementioned plasmid, wherein the DNA sequence encoding the rRNA geneis under the control of an inducible promoter.

Another aspect of the invention relates to a cell comprising anaforementioned plasmid. In certain embodiments said cell comprises aplasmid which comprises a first nucleic acid sequence and a secondnucleic acid sequence; wherein said first nucleic acid sequence encodesa Pseudomonas aeruginosa 16S rRNA comprising a mutantAnti-Shine-Dalgarno sequence and at least one additional mutationoutside the Anti-Shine-Dalgarno region; and said second sequence encodesa selectable marker having a mutant Shine-Dalgarno sequence; whereinsaid mutant Anti-Shine-Dalgarno specifically hybridizes to said mutantShine-Dalgarno sequence. In certain embodiments said cell comprises aplasmid which comprises a first nucleic acid sequence and a secondnucleic acid sequence; wherein said first nucleic acid sequence encodesa Pseudomonas aeruginosa 16S rRNA comprising a mutantAnti-Shine-Dalgarno sequence and at least one additional mutationoutside the Anti-Shine-Dalgarno region; and said second sequence encodesa selectable marker having a mutant Shine-Dalgarno sequence; whereinsaid mutant Anti-Shine-Dalgarno specifically hybridizes to said mutantShine-Dalgarno sequence.

Another aspect of the invention relates to a cell comprising two of theaforementioned plasmids. In certain embodiments said cell comprises aplasmid which comprises a first nucleic acid sequence and a secondnucleic acid sequence; wherein said first nucleic acid sequence encodesa Pseudomonas aeruginosa 16S rRNA comprising a mutantAnti-Shine-Dalgarno sequence and at least one additional mutationoutside the Anti-Shine-Dalgarno region; and said second sequence encodesa selectable marker having a mutant Shine-Dalgarno sequence; whereinsaid mutant Anti-Shine-Dalgarno specifically hybridizes to said mutantShine-Dalgarno sequence; and a plasmid which comprises a Pseudomonasaeruginosa S20 gene.

In certain embodiments the present invention relates to theaforementioned cell, wherein the mutations in the rRNA gene affect thequantity of selectable marker produced.

In certain embodiments the present invention relates to theaforementioned cell, wherein the cell is a bacterial cell.

In certain embodiments the present invention relates to theaforementioned cell, wherein the cell is an E. coli cell.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes comprising:

-   -   (a) transforming a set of host cells with a set of plasmids,        each plasmid comprising a mutant Pseudomonas aeruginosa 16S rRNA        gene and a selectable marker gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation and a first mutant            Anti-Shine-Dalgarno sequence; and said first selectable            marker gene comprises a first mutant Shine-Dalgarno            sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a set of transformed host cells;    -   (b) transforming said set of host cells with a plasmid        comprising a Pseudomonas aeruginosa S20 gene;    -   (c) isolating from the set of transformed host cells those host        cells which express the selectable marker gene product; and    -   (d) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (c), thereby identifying        functional mutant ribosomes.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes comprising:

-   -   (a) transforming a set of host cells with a set of plasmids,        each plasmid comprising a mutant Pseudomonas aeruginosa 16S rRNA        gene and a green fluorescent protein gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation and a first mutant            Anti-Shine-Dalgarno sequence; and said green fluorescent            protein gene comprises a first mutant Shine-Dalgarno            sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a set of transformed host cells;    -   (b) transforming said set of host cells with a plasmid        comprising a Pseudomonas aeruginosa S20 gene;    -   (c) isolating from the set of transformed host cells those host        cells which express the green fluorescent protein gene product;        and    -   (d) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (c), thereby identifying        functional mutant ribosomes.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes that may be suitable as drug targetscomprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a selectable marker gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation and a first mutant            Anti-Shine-Dalgarno sequence; and said first selectable            marker gene comprises a first mutant Shine-Dalgarno            sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) transforming said first set of host cells with a plasmid        comprising a Pseudomonas aeruginosa S20 gene;    -   (c) isolating from the first set of transformed host cells those        host cells which express the selectable marker gene product;    -   (d) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (c) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (e) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (d) are        mutated; and each rRNA gene further comprises a second mutant        Anti-Shine-Dalgarno sequence;    -   (f) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (e) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second selectable marker having a second mutant        Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (g) transforming a second set of host cells with the plasmids        from step (f) and plasmids comprising a Pseudomonas aeruginosa        S20 gene, thereby forming a second set of transformed host        cells;    -   (h) isolating from the second set of transformed host cells from        step (g) those host cells which express the selectable marker        gene product; and    -   (i) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (h), thereby identifying        functional mutant ribosomes that may be suitable as drug        targets.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes that may be suitable as drug targetscomprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a first green fluorescent protein        gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation and a first mutant            Anti-Shine-Dalgarno sequence; and said first green            fluorescent protein gene comprises a first mutant            Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) transforming said first set of host cells with a plasmid        comprising a Pseudomonas aeruginosa S20 gene;    -   (c) isolating from the first set of transformed host cells those        host cells which express the green fluorescent protein gene        product;    -   (d) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (c) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (e) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (d) are        mutated; and each rRNA gene further comprises a second mutant        Anti-Shine-Dalgarno sequence;    -   (f) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (e) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second green fluorescent protein having a second        mutant Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (g) transforming a second set of host cells with the plasmids        from step (f) and plasmids comprising a Pseudomonas aeruginosa        S20 gene, thereby forming a second set of transformed host        cells;    -   (h) isolating from the second set of transformed host cells from        step (g) those host cells which express the green fluorescent        protein gene product; and    -   (i) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (h), thereby identifying        functional mutant ribosomes that may be suitable as drug        targets.

Another aspect of the present invention is a method for identifying drugcandidates comprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a selectable marker gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation and a first mutant            Anti-Shine-Dalgarno sequence; and said first selectable            marker gene comprises a first mutant Shine-Dalgarno            sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) transforming said first set of host cells with a plasmid        comprising a Pseudomonas aeruginosa S20 gene;    -   (c) isolating from the first set of transformed host cells those        host cells which express the selectable marker gene product;    -   (d) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (c) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (e) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (d) are        mutated; and each rRNA gene further comprises a second mutant        Anti-Shine-Dalgarno sequence;    -   (f) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (e) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second selectable marker having a second mutant        Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (g) transforming a second set of host cells with the plasmids        from step (f) and plasmids comprising a Pseudomonas aeruginosa        S20 gene, thereby forming a second set of transformed host        cells;    -   (h) isolating from the second set of transformed host cells from        step (g) those host cells which express the selectable marker        gene product;    -   (i) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (h), to identify the mutated        regions of interest;    -   (j) screening compounds against the mutated regions of interest        from step (i) and wildtype Pseudomonas aeruginosa 16S rRNA;    -   (k) identifying the compounds from step (j) that bind to the        mutated regions of interest from step (h) and the wildtype        Pseudomonas aeruginosa 16S rRNA;    -   (l) screening the compounds from step (k) against human 16S        rRNA; and    -   (m) identifying the drug candidates from step (1) that do not        bind to the human 16S rRNA, thereby identifying drug candidates.

Another aspect of the present invention is a method for identifying drugcandidates comprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a first green fluorescent protein        gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation and a first mutant            Anti-Shine-Dalgarno sequence; and said first green            fluorescent protein gene comprises a first mutant            Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) transforming said first set of host cells with a plasmid        comprising a Pseudomonas aeruginosa S20 gene;    -   (c) isolating from the first set of transformed host cells those        host cells which express the green fluorescent protein gene        product;    -   (d) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (c) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (e) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (d) are        mutated; and each rRNA gene further comprises a second mutant        Anti-Shine-Dalgarno sequence;    -   (f) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (e) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second green fluorescent protein having a second        mutant Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (g) transforming a second set of host cells with the plasmids        from step (f) and plasmids comprising a Pseudomonas aeruginosa        S20 gene, thereby forming a second set of transformed host        cells;    -   (h) isolating from the second set of transformed host cells from        step (g) those host cells which express the green fluorescent        protein gene product;    -   (i) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (h), to identify the mutated        regions of interest;    -   (j) screening compounds against the mutated regions of interest        from step (i) and wildtype Pseudomonas aeruginosa 16S rRNA;    -   (k) identifying the compounds from step (j) that bind to the        mutated regions of interest from step (h) and the wildtype        Pseudomonas aeruginosa 16S rRNA;    -   (l) screening the compounds from step (k) against human 16S        rRNA; and    -   (m) identifying the drug candidates from step (l) that do not        bind to the human 16S rRNA, thereby identifying drug candidates.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes comprising:

-   -   (a) transforming a set of host cells with a set of plasmids,        each plasmid comprising a mutant Pseudomonas aeruginosa 16S rRNA        gene and a selectable marker gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation, a mutant helix 9 sequence,            and a first mutant Anti-Shine-Dalgarno sequence; and said            first selectable marker gene comprises a first mutant            Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a set of transformed host cells;    -   (b) isolating from the set of transformed host cells those host        cells which express the selectable marker gene product; and    -   (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (b), thereby identifying        functional mutant ribosomes.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes comprising:

-   -   (a) transforming a set of host cells with a set of plasmids,        each plasmid comprising a mutant Pseudomonas aeruginosa 16S rRNA        gene and a green fluorescent protein gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation, a mutant helix 9 sequence,            and a first mutant Anti-Shine-Dalgarno sequence; and said            green fluorescent protein gene comprises a first mutant            Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a set of transformed host cells;    -   (b) isolating from the set of transformed host cells those host        cells which express the green fluorescent protein gene product;        and    -   (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (b), thereby identifying        functional mutant ribosomes.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes that may be suitable as drug targetscomprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a selectable marker gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation, a mutant helix 9 sequence,            and a first mutant Anti-Shine-Dalgarno sequence; and said            first selectable marker gene comprises a first mutant            Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) isolating from the first set of transformed host cells those        host cells which express the selectable marker gene product;    -   (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (b) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (d) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (c) are        mutated; and each mutant Pseudomonas aeruginosa 16S rRNA gene        further comprises a mutant helix 9 sequence, and a second mutant        Anti-Shine-Dalgarno sequence;    -   (e) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (d) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second selectable marker having a second mutant        Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (f) transforming a second set of host cells with the plasmids        from step (e), thereby forming a second set of transformed host        cells;    -   (g) isolating from the second set of transformed host cells from        step (f) those host cells which express the selectable marker        gene product; and    -   (h) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (g), thereby identifying        functional mutant ribosomes that may be suitable as drug        targets.

Another aspect of the present invention is a method for identifyingfunctional mutant ribosomes that may be suitable as drug targetscomprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a first green fluorescent protein        gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation, a mutant helix 9 sequence,            and a first mutant Anti-Shine-Dalgarno sequence; and said            first green fluorescent protein gene comprises a first            mutant Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) isolating from the first set of transformed host cells those        host cells which express the green fluorescent protein gene        product;    -   (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (b) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (d) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (d) are        mutated; and each mutant Pseudomonas aeruginosa 16S rRNA gene        further comprises a mutant helix 9 sequence, and a second mutant        Anti-Shine-Dalgarno sequence;    -   (e) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (d) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second green fluorescent protein having a second        mutant Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (f) transforming a second set of host cells with the plasmids        from step (e), thereby forming a second set of transformed host        cells;    -   (g) isolating from the second set of transformed host cells from        step (f) those host cells which express the green fluorescent        protein gene product; and    -   (h) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (g), thereby identifying        functional mutant ribosomes that may be suitable as drug        targets.

Another aspect of the present invention is a method for identifying drugcandidates comprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a selectable marker gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation, a mutant helix 9 sequence,            and a first mutant Anti-Shine-Dalgarno sequence; and said            first selectable marker gene comprises a first mutant            Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) isolating from the first set of transformed host cells those        host cells which express the selectable marker gene product;    -   (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (b) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (d) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (c) are        mutated; and each mutant Pseudomonas aeruginosa 16S rRNA gene        further comprises a mutant helix 9 sequence, and a second mutant        Anti-Shine-Dalgarno sequence;    -   (e) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (d) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second selectable marker having a second mutant        Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (f) transforming a second set of host cells with the plasmids        from step (e), thereby forming a second set of transformed host        cells;    -   (g) isolating from the second set of transformed host cells from        step (f) those host cells which express the selectable marker        gene product;    -   (h) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (g), to identify the mutated        regions of interest;    -   (i) screening compounds against the mutated regions of interest        from step (h) and wildtype Pseudomonas aeruginosa 16S rRNA;    -   (j) identifying the compounds from step (i) that bind to the        mutated regions of interest from step (h) and the wildtype        Pseudomonas aeruginosa 16S rRNA;    -   (k) screening the compounds from step (j) against human 16S        rRNA; and    -   (l) identifying the drug candidates from step (k) that do not        bind to the human 16S rRNA, thereby identifying drug candidates.

Another aspect of the present invention is a method for identifying drugcandidates comprising:

-   -   (a) transforming a first set of host cells with a first set of        plasmids, each plasmid comprising a mutant Pseudomonas        aeruginosa 16S rRNA gene and a first green fluorescent protein        gene;        -   wherein said mutant Pseudomonas aeruginosa 16S rRNA gene            comprises at least one mutation, a mutant helix 9 sequence,            and a first mutant Anti-Shine-Dalgarno sequence; and said            first green fluorescent protein gene comprises a first            mutant Shine-Dalgarno sequence; and        -   wherein said first mutant Anti-Shine-Dalgarno sequence and            said first mutant Shine-Dalgarno sequence are a mutually            compatible pair;        -   thereby forming a first set of transformed host cells;    -   (b) isolating from the first set of transformed host cells those        host cells which express the green fluorescent protein gene        product;    -   (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene        from each host cell isolated in step (b) to identify regions of        interest, wherein the regions of interest comprise sequences of        one or more nucleic acids which are conserved in each first        mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;    -   (d) generating a second set of mutant Pseudomonas aeruginosa 16S        rRNA genes wherein the regions of interest from step (d) are        mutated; and each mutant Pseudomonas aeruginosa 16S rRNA gene        further comprises a mutant helix 9 sequence, and a second mutant        Anti-Shine-Dalgarno sequence;    -   (e) inserting the second set of mutant Pseudomonas aeruginosa        16S rRNA genes comprising the mutated regions of interest from        step (d) into a second set of plasmids; wherein said plasmids        further comprise a second genetically engineered gene which        encodes a second green fluorescent protein having a second        mutant Shine-Dalgarno sequence, wherein the second mutant        Anti-Shine-Dalgarno and the second mutant Shine-Dalgarno        sequence are a mutually compatible pair;    -   (f) transforming a second set of host cells with the plasmids        from step (e), thereby forming a second set of transformed host        cells;    -   (g) isolating from the second set of transformed host cells from        step (f) those host cells which express the green fluorescent        protein gene product;    -   (h) sequencing the Pseudomonas aeruginosa 16S rRNA gene from        each host cell isolated in step (g), to identify the mutated        regions of interest;    -   (i) screening compounds against the mutated regions of interest        from step (h) and wildtype Pseudomonas aeruginosa 16S rRNA;    -   (j) identifying the compounds from step (i) that bind to the        mutated regions of interest from step (h) and the wildtype        Pseudomonas aeruginosa 16S rRNA;    -   (k) screening the compounds from step (j) against human 16S        rRNA; and    -   (l) identifying the drug candidates from step (k) that do not        bind to the human 16S rRNA, thereby identifying drug candidates.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and Appendices, are incorporatedherein by reference.

EXEMPLIFICATION Example 1 Identification of Mutant SD AND Mutant ASDCombinations

It has been shown that by coordinately changing the SD and ASD, aparticular mRNA containing an altered SD could be targeted to ribosomescontaining the altered ASD. This and all other efforts to modify theASD, however, have proved lethal, as cells containing these mutationsdied within two hours after the genes containing them were activated.

Using random mutagenesis and genetic selection, mutant SD-ASDcombinations are screened in order to identify non-lethal SD-ASDcombinations. The mutant SD-ASD mutually compatible pairs are set forthin FIGS. 10, 11 and 12. The mutually compatible pairs of mutantsequences interact as pairs in the form of RNA. The novel mutant SD-ASDsequence combinations of the present invention permit translation ofonly the mRNAs containing the altered SD sequence.

Example 2A Construction of the Pa 16S pRNA228 Plasmid

A plasmid construct of the present invention identified as the Pa 16SpRNA228 plasmid, is set forth in FIGS. 1 and 6. E. coli cells contain asingle chromosome with seven copies of the rRNA genes and all of thegenes for the ribosomal proteins. The plasmid, Pa 16S pRNA228, in thecell contains a genetically engineered copy of one of the 16S rRNA genesfrom P. aeruginosa and two genetically engineered genes that are notnormally found in E. coli, referred to herein as a “selectable markers.”One gene encodes the protein chloramphenicol acetyltransferase (CAT).This protein renders cells resistant to chloramphenicol by chemicallymodifying the antibiotic. Another gene, the Green Fluorescent Protein(GFP), is also included in the system. GFP facilitates high-throughputfunctional analysis. The amount of green light produced upon irradiationwith ultraviolet light is proportional to the amount of GFP present inthe cell.

Ribosomes from Pa 16S pRNA228 have an altered ASD sequence. Therefore,the ribosomes can only translate mRNAs that have an altered SD sequence.Only two genes in the cell produce mRNAs with altered SD sequences thatmay be translated by the plasmid-encoded ribosomes: the CAT and GFPgene. Mutations in rRNA affect the ability of the resulting mutantribosome to make protein. The present invention thus provides a systemwhereby the mutations in the plasmid-encoded rRNA gene only affect theamount of GFP and CAT produced. A decrease in plasmid ribosome functionmakes the cell more sensitive to chloramphenicol and reduces the amountof green fluorescence of the cells. Translation of the other mRNAs inthe cell is unaffected since these mRNAs are translated only byribosomes that come from the chromosome. Hence, cells containingfunctional mutants may be identified and isolated via the selectablemarker.

Example 2B Construction of the Pa 16S pRNA228 Plasmid

A plasmid construct of the present invention identified as the Pa 16S EcH9 pRNA228 plasmid, is set forth in FIGS. 2 and 7. E. coli cells containa single chromosome with seven copies of the rRNA genes and all of thegenes for the ribosomal proteins. The plasmid, Pa 16S Ec H9 pRNA228, inthe cell contains a genetically engineered copy of one of the 16S rRNAgenes from P. aeruginosa wherein the P. aeruginosa helix 9 sequence hasbeen replaced with the corresponding E. coli sequence, and twogenetically engineered genes that are not normally found in E. coli,referred to herein as a “selectable markers.” One gene encodes theprotein chloramphenicol acetyltransferase (CAT). This protein renderscells resistant to chloramphenicol by chemically modifying theantibiotic. Another gene, the Green Fluorescent Protein (GFP), is alsoincluded in the system. GFP facilitates high-throughput functionalanalysis. The amount of green light produced upon irradiation withultraviolet light is proportional to the amount of GFP present in thecell.

Ribosomes from Pa 16S Ec H9 pRNA228 have an altered ASD sequence.Therefore, the ribosomes can only translate mRNAs that have an alteredSD sequence. Only two genes in the cell produce mRNAs with altered SDsequences that may be translated by the plasmid-encoded ribosomes: theCAT and GFP gene. Mutations in rRNA affect the ability of the resultingmutant ribosome to make protein. The present invention thus provides asystem whereby the mutations in the plasmid-encoded rRNA gene onlyaffect the amount of GFP and CAT produced. A decrease in plasmidribosome function makes the cell more sensitive to chloramphenicol andreduces the amount of green fluorescence of the cells. Translation ofthe other mRNAs in the cell is unaffected since these mRNAs aretranslated only by ribosomes that come from the chromosome. Hence, cellscontaining functional mutants may be identified and isolated via theselectable marker.

Example 2C Construction of the pKan5-T1T2 and pKanPa-S20 Plasmid

A plasmid construct of the present invention identified as thepKan5-T1T2 plasmid, is set forth in FIGS. 3 and 8. The plasmid,pKan5-T1T2, is a PACYC177 derivative. It was used in the preparation ofthe pKanPa-S20 plasmid, which encodes the P. aeruginosa S20 protein,which is set forth in FIGS. 4 and 9.

Example 3 Genetic System for Functional Analysis of Ribosomal RNA

Identification of Functionally Important Regions of rRNA. Functionallyimportant regions of rRNA molecules that may be used as drug targetsusing a functional genomics approach may be identified through a seriesof steps. Namely, in step I.a., the entire rRNA gene is randomly mutatedusing error-prone PCR or another generalized mutational strategy. Instep I.b., a host cell is then transformed with a mutagenized plasmidcomprising: an rRNA gene having a mutant ASD sequence, at least onemutation in said rRNA gene, and a genetically engineered gene whichencodes a selectable marker having a mutant SD sequence, and productionof the rRNA genes from the plasmid are induced by growing the cells inthe presence of IPTG. In step I.c., the CAT gene is used to selectmutants that are functional by plating the transformed cells onto growthmedium containing chloramphenicol. In step I.d., individual clones fromeach of the colonies obtained in step I.c. are isolated. In step I.e.,the plasmid DNA from each of the individual clones from step I.d. isisolated. In step I.f., the rRNA genes contained in each of the plasmidsthat had been isolated in step I.e. are sequenced. In step I.g., thefunction of each of the mutants from step I.f. is assessed by measuringthe amount of GFP produced by each clone from step I.e. upon inductionof the rRNA operon. In step I.h., a functional genomics database isassembled containing the sequence and functional data from steps I.f.and I.g. In step I.i., functionally important regions of the rRNA genethat will serve as drug targets are identified. Functionally importantregions may be identified by comparing the sequences of all of thefunctional genomics database constructed in step I.g. and correlatingthe sequence with the amount of GFP protein produced. Contiguoussequences of three or more rRNA nucleotides, in which substitution ofthe nucleotides in the region produces significant loss of function,will constitute a functionally important region and therefore apotential drug target. Helix 9 (SEQ ID NO 1 and SEQ ID NO 2) is afunctional important region, as shown herein, and is therefore apotential drug target.

Isolation of Functional Variants of the Target Regions. A second aspectof the invention features identification of mutations of the target sitethat might lead to antibiotic resistance using a process termed,“instant evolution”, as described below. In step II.a., for a giventarget region identified in step I.i., each of the nucleotides in thetarget region is simultaneously randomly mutated using standard methodsof molecular mutagenesis, such as cassette mutagenesis or PCRmutagenesis, and cloned into the plasmid of step I.b. to form a plasmidpool containing random mutations at each of the nucleotide positions inthe target region. In step II.b., the resulting pool of plasmidscontaining random mutations from step II.a. is used to transform E. colicells and form a library of clones, each of which contains a uniquecombination of mutations in the target region. In step II.c., thelibrary of mutant clones from step II.b. is grown in the presence ofIPTG to induce production of the mutant rRNA genes. In step II.d., theinduced mutants are plated on medium containing chloramphenicol, and CATis used to select clones of rRNA mutants containing nucleotidecombinations of the target region that produce functional ribosomes. Instep II.e., the functional clones isolated in step II.d. are sequencedand GFP is used to measure ribosome function in each one. In step II.f.,the data from step II.e. are incorporated into a mutational database.

Isolation of Drug Leads. In step III.a., the database in step II.f. isanalyzed to identify functionally-important nucleotides and nucleotidemotifs within the target region. In step III.b., the information fromstep III.a. is used to synthesize a series of oligonucleotides thatcontain the functionally important nucleotides and nucleotide motifsidentified in step III.a. In step III.c., the oligonucleotides from stepIII.b. are used to sequentially screen compounds and compound librariesto identify compounds that recognize (bind to) the functionallyimportant sequences and motifs. In step III.d., compounds that bind toall of the oligonucleotides are counterscreened against oligonucleotidesand/or other RNA containing molecules to identify drug candidates. “Drugcandidates” are compounds that 1) bind to all of the oligonucleotidescontaining the functionally important nucleotides and nucleotide motifs,but do not bind to molecules that do not contain the functionallyimportant nucleotides and nucleotide motifs and 2) do not recognizehuman ribosomes. Drug candidates selected by the methods of the presentinvention therefore recognize all of the functional variants of thetarget sequence, i.e., the target cannot be mutated in a way that thedrug cannot bind, without causing loss of function to the ribosome.

Example 4 Genetic System for Studying Protein Synthesis

All plasmids are maintained and expressed in E. coli DH5 (e.g. supE44,hsdR17, recA1, endA1, gyrA96, thi-1 and relA1; Hanahan, D. (1983) J.Mol. Biol. 166:557-580). To induce synthesis of plasmid-derived rRNAfrom the lacUV5 promoter, IPTG is added to a final concentration of 1mM. Chloramphenicol acetyltransferase activity will be determinedessentially as described by Nielsen et al. (1989) Anal. Biochem. 179:19-23. Cultures for CAT assays are grown in LB-Ap100. MIC will bedetermined by standard methods in microtiter plates as described in Lee,K., et al. (1997) J. Mol. Biol. 269: 732-743. Procedures are followed asin outlined in Example 4 of Cunningham et al. (WO 2004/003511).

Example 5 In Vivo Determination of RNA Structure-Function Relationships

Bacterial strains and media. Plasmids are maintained and expressed in E.coli DH5 (e.g. supE44, hsdR17, recA1, endA1, gyrA9 and thi-1; Hanahan,D. (1983) J. Mol. Biol. 166:557-580). Cultures are grown in LB medium(Luria, S. E. & Burrous, J. W. (1957) J. Bacteriol. 74:461-476) or LBmedium containing 100 μg/ml ampicillin (LB-Ap100). To induce synthesisof plasmid-derived rRNA from the lacUV5 promoter, IPTG is added to afinal concentration of 1 mM at the times indicated in each experiment.Strains are transformed by electroporation (Dower, W. J., et al. (1988)Nucl. Acids Res. 16: 6127) using a Gibco-BRL Cell Porator. Transformantsare grown in SOC medium (Hanahan, 1983, supra) for one hour prior toplating on selective medium to allow expression of plasmid-derivedgenes.

Chloramphenicol acetyltransferase assays. CAT activity is determinedessentially as described (Nielsen, D. A. et al. (1989) Anal. Biochem.60:191-227). Cultures for CAT assays will begrown in LB-Ap100. Briefly,0.5 ml aliquots of mid-log cultures (unless otherwise indicated) isadded to an equal volume of 500 mM Tris-HCl (pH8) and lysed using 0.01%(w/v) SDS and chloroform (Miller, J. H. (1992) A Short Course inBacterial Genetics, (Miller, J. H., ed.), pp. 71-80, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The resulting lysate iseither be used directly or diluted in assay buffer prior to use. Assaymixtures contain cell extract (5 μl or 10 μl ), 250 mM Tris (pH 8), 214μM butyryl-coenzyme A (Bu-CoA), and 40 μM [H]chloramphenicol in a 125 μlvolume. Two concentrations of lysate are assayed for one hour at 37° C.to ensure that the signal was proportional to protein concentrations.The product, butyryl-[³ H]chloramphenicol is extracted into2,6,10,14-tetramethylpentadecane:xylenes (2:1) and measured directly ina Beckman LS-3801 liquid scintillation counter. Blanks are preparedexactly as described above, except that uninoculated LB medium was usedinstead of culture.

Minimum inhibitory concentration determination. MICs are determined bystandard methods in microtiter plates or on solid medium. Overnightcultures grown in LB-Ap100 are diluted and induced in the same mediumcontaining 1 mM IPTG for three hours. Approximately 10 ⁴ induced cellsare then added to wells (or spotted onto solid medium) containingLB-Ap100 +IPTG (1 mM) and chloramphenicol at increasing concentrations.Cultures are grown for 24 hours and the lowest concentration ofchloramphenicol that completely inhibited growth is designated as theMIC.

Oligoribonucleotide synthesis. Oligoribonucleotides are synthesized onsolid support with the phosphoramidite method (Capaldi, D. & Reese, C.(1994) Nucl. Acids Res. 22:2209-2216) on a Cruachem PS 250 DNA/RNAsynthesizer. Oligomers are removed from solid support and deprotected bytreatment with ammonia and acid following the manufacturer'srecommendations. The RNA is purified on a silica gel Si500F TLC plate(Baker) eluted for five hours with n-propanol/ammonia/water (55:35:10,by vol.). Bands are visualized with an ultraviolet lamp and the leastmobile band was cut out and eluted three times with 1 ml of purifiedwater. Oligomers are further purified with a Sep-pak C-18 cartridge(Waters) and desalted by continuous-flow dialysis (BRL). Purities arechecked by analytical C-8 HPLC (Perceptive Biosystems).

Incorporation By Reference

All of the references including, without limitation, U.S. patents, U.S.patent application publications, published international applicationsand journal articles cited herein are hereby incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

I claim:
 1. A method for identifying functional mutant ribosomescomprising: (a) transforming a set of host cells with a set of plasmids,each plasmid comprising a mutant Pseudomonas aeruginosa 16S rRNA geneand a selectable marker gene; wherein said mutant Pseudomonas aeruginosa16S rRNA gene comprises at least one mutation, and a mutant helix 9sequence, in addition to a first mutant Anti-Shine-Dalgarno sequence;and said first selectable marker gene comprises a first mutantShine-Dalgarno sequence; and wherein said first mutantAnti-Shine-Dalgarno sequence and said first mutant Shine-Dalgarnosequence are a mutually compatible pair; thereby forming a set oftransformed host cells; (b) isolating from the set of transformed hostcells those host cells which express the selectable marker gene product;and (c) sequencing the mutant Pseudomonas aeruginosa 16S rRNA gene fromeach host cell isolated in step (b), thereby identifying functionalmutant ribosomes.
 2. The method of claim 1, wherein the selectablemarker gene is a green fluorescent protein gene.
 3. The method of claim1, wherein said mutant helix 9 sequence is SEQ ID NO:
 1. 4. The methodof claim 1, wherein the mutant Anti-Shine-Dalgarno sequence is selectedfrom the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 and
 148. 5. Themethod of claim 1, wherein the mutant Shine-Dalgarno sequence isselected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 and 147.6. The method of claim 1, wherein the mutant Shine-Dalgarno sequence andthe mutant Anti-Shine-Dalgarno sequence are a complementary pairselected from the group consisting of SEQ ID NOs: 7 and 8, 9 and 10, 11and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112,113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and124, 125 and 126, 127 and 128, 129 and 130, 131 and 132, 133 and 134,135 and 136, 137 and 138, 139 and 140, 141 and 142, 143 and 144, 145 and146, and 147 and
 148. 7. The method of claim 1, wherein the functionalmutant ribosomes of step (c) have regions of interest which comprisesequences of one or more nucleic acids which are conserved in eachmutant Pseudomonas aeruginosa 16S rRNA gene sequenced; and the methodfurther comprising: (d) generating a second set of mutant Pseudomonasaeruginosa 16S rRNA genes wherein the regions of interest from step (c)are mutated; and each mutant Pseudomonas aeruginosa 16S rRNA genefurther comprises a mutant helix 9 sequence, and a second mutantAnti-Shine-Dalgarno sequence; (e) inserting the second set of mutantPseudomonas aeruginosa 16S rRNA genes comprising the mutated regions ofinterest from step (d) into a second set of plasmids; wherein saidplasmids further comprise a second genetically engineered gene whichencodes a second selectable marker having a second mutant Shine-Dalgarnosequence, wherein the second mutant Anti-Shine-Dalgarno and the secondmutant Shine-Dalgarno sequence are a mutually compatible pair; (f)transforming a second set of host cells with the plasmids from step (e),thereby forming a second set of transformed host cells; (g) isolatingfrom the second set of transformed host cells from step (f) those hostcells which express the selectable marker gene product; and (h)sequencing the Pseudomonas aeruginosa 16S rRNA gene from each host cellisolated in step (g), thereby identifying functional mutant ribosomesthat may be suitable as drug targets.
 8. The method of claim 7, whereinthe selectable marker gene is a green fluorescent protein gene, and thesecond selectable marker is a green fluorescent protein.
 9. The methodof claim 7, wherein said mutant helix 9 sequence is SEQ ID NO:
 1. 10.The method of claim 7, wherein the mutant Anti-Shine-Dalgarno sequenceand the second mutant Anti-Shine-Dalgarno sequence are selected from thegroup consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146 and
 148. 11. The methodof claim 7, wherein the mutant Shine-Dalgarno sequence and the secondmutant Shine-Dalgarno sequence are each selected from the groupconsisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145 and
 147. 12. The method of claim7, wherein the mutant Shine-Dalgarno sequence and the mutantAnti-Shine-Dalgarno sequence and the second mutant Shine-Dalgarnosequence and the second mutant Anti-Shine-Dalgarno sequence are each acomplementary pair selected from the group consisting of SEQ ID NOs: 7and 8, 9 and 10, 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20,21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120,121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, 131 and132, 133 and 134, 135 and 136, 137 and 138, 139 and 140, 141 and 142,143 and 144, 145 and 146, and 147 and
 148. 13. The method of claim 1,wherein the functional mutant ribosomes of step (c) have regions ofinterest which comprise sequences of one or more nucleic acids which areconserved in each mutant Pseudomonas aeruginosa 16S rRNA gene sequenced;and the method further comprising: (d) generating a second set of mutantPseudomonas aeruginosa 16S rRNA genes wherein the regions of interestfrom step (c) are mutated; and each mutant Pseudomonas aeruginosa 16SrRNA gene further comprises a mutant helix 9 sequence, and a secondmutant Anti-Shine-Dalgarno sequence; (e) inserting the second set ofmutant Pseudomonas aeruginosa 16S rRNA genes comprising the mutatedregions of interest from step (d) into a second set of plasmids; whereinsaid plasmids further comprise a second genetically engineered genewhich encodes a second selectable marker having a second mutantShine-Dalgarno sequence, wherein the second mutant Anti-Shine-Dalgarnoand the second mutant Shine-Dalgarno sequence are a mutually compatiblepair; (f) transforming a second set of host cells with the plasmids fromstep (e), thereby forming a second set of transformed host cells; (g)isolating from the second set of transformed host cells from step (f)those host cells which express the selectable marker gene product; (h)sequencing the Pseudomonas aeruginosa 16S rRNA gene from each host cellisolated in step (g), to identify the mutated regions of interest; (i)screening compounds against the mutated regions of interest from step(h) and wildtype Pseudomonas aeruginosa 16S rRNA; (j) identifying thecompounds from step (i) that bind to the mutated regions of interestfrom step (h) and the wildtype Pseudomonas aeruginosa 16S rRNA; (k)screening the compounds from step (j) against human 16S rRNA; and (1)identifying the drug candidates from step (k) that do not bind to thehuman 16S rRNA, thereby identifying drug candidates.
 14. The method ofclaim 13, wherein the selectable marker gene is a green fluorescentprotein gene, and the second selectable marker is a green fluorescentprotein.
 15. The method of claim 13, wherein said mutant helix 9sequence is SEQ ID NO:
 1. 16. The method of claim 13, wherein the mutantAnti-Shine-Dalgarno sequence and the second mutant Anti-Shine-Dalgarnosequence are selected from the group consisting of SEQ ID NOs: 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146 and
 148. 17. The method of claim 13, wherein the mutantShine-Dalgarno sequence and the second mutant Shine-Dalgarno sequenceare each selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145 and147.
 18. The method of claim 13, wherein the mutant Shine-Dalgarnosequence and the mutant Anti-Shine-Dalgarno sequence and the secondmutant Shine-Dalgarno sequence and the second mutant Anti-Shine-Dalgarnosequence are each a complementary pair selected from the groupconsisting of SEQ ID NOs: 7 and 8, 9 and 10, 11 and 12, 13 and 14, 15and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126,127 and 128, 129 and 130, 131 and 132, 133 and 134, 135 and 136, 137 and138, 139 and 140, 141 and 142, 143 and 144, 145 and 146, and 147 and148.